Abstract This competitive renewal application seeks to investigate the role of translesion synthesis DNA polymerases in mutagenesis associated with meiotic cell divisions. Unlike somatic mutations that only impact the individual, DNA changes occurring during meiosis are transmitted to the next generations and lead to the development of hereditary diseases. With the childbirth age steadily increasing over the past decades, the frequency of de novo germline mutations in the human population has continuously been on the rise. The resulting hereditary diseases present a significant burden for the individuals, families and the society, since the patients often develop multiple medical problems at an early age and require specialized life-long care. The mechanisms responsible for the generation of germline mutations are poorly understood, as nearly all mechanistic studies of mutagenesis employ mitotic cells. In the previous cycles of this grant, we discovered and explored a novel mutagenesis pathway wherein error-prone DNA polymerase ? (Pol?) is recruited to DNA replication forks stalled at small hairpin DNA structures and facilitates the bypass of these structures, producing a characteristic mutational signature. This pathway is silent in healthy mitotic cells because the robust normal replication machinery is not significantly impeded by the small secondary structures. The Pol?-dependent error-prone structure bypass, however, becomes a factor when intrinsic or environmental stressors promote fork stalling. Unexpectedly, our preliminary data suggested that this pathway is also activated during normal meiosis and is a likely source of recurrent germline mutations in cancer predisposition genes. We will test this hypothesis by pursuing three Specific Aims. In Aim 1, we will define the contribution of the Pol?-dependent pathway to germline- and meiosis-specific mutagenesis. In Aim 2, we will use the human POLE gene linked to a hereditary colorectal cancer predisposition syndrome as a model to identify meiosis-specific hotspots of mutagenesis and define their relationship to Pol?-dependent hairpin bypass. In Aim 3, we will determine the effects of environmental DNA damaging agents and replication inhibitors on the accumulation of Pol?-dependent mutations during gametogenesis. Yeast, mouse and human cell models will be used in these studies, with the yeast system providing the most power for mechanistic analysis, the mouse providing the opportunity to experimentally study mutagenesis during mammalian meiosis in vivo, and the data on human samples establishing the ultimate link to disease. We expect to gain new fundamental knowledge on the mechanism of mutagenesis in the germline that impacts future generations. We also expect to understand the reasons for frequent de novo formation of some disease-causing germline variants and the effects of environmental factors.